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Transcriptomic analyses of cardiomyopathy development in mice carrying the phospholamban p.(Arg14del) pathogenic variant, and comparison with ischemic heart disease

ABSTRACT: Background: The p.(Arg14del) pathogenic variant (R14del) of the phospholamban (PLN) gene is a prevalent cause of cardiomyopathy with heart failure. The exact underlying pathophysiology is unknown, and a suitable therapy is unavailable. We aim to identify molecular perturbations underlying this cardiomyopathy in a clinically relevant PLN-R14del mouse model. Methods: We investigated progression of cardiomyopathy in PLN-R14∆/∆ mice using echocardiography, electrocardiography and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 weeks of age (before onset of disease), 5 weeks (mild cardiomyopathy), and 8 weeks (end-stage). Data were compared with cardiac expression levels of mice that underwent myocardial ischemia-reperfusion or myocardial infarction surgery, in an effort to identify alterations that are specific to PLN-R14del-related cardiomyopathy. Results: At 3 weeks of age, PLN-R14∆/∆ mice had normal cardiac function, but from the age of 4 weeks, we observed increased myocardial fibrosis and impaired global longitudinal strain. From 5 weeks onwards, ventricular dilatation, decreased contractility and diminished electrocardiogram voltages were observed. Strikingly, PLN protein aggregation was present prior to onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodeling, inflammation and metabolic dysfunction, in part similar to ischemic cardiomyopathy. Altered protein homeostasis pathways were identified exclusively in PLN-R14∆/∆ mice, even before disease onset, in concert with aggregate formation. Conclusions: We mapped the development of PLN-R14del-related cardiomyopathy, and identified alterations in proteostasis and PLN protein aggregation amongst the first manifestations of this disease, which could possibly be a novel target for therapy.

ORGANISM(S): Mus musculus

PROVIDER: GSE168610 | GEO | 2021/07/22

REPOSITORIES: GEO

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Project description:Aims: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and Results: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3±1.2 Hz in hPLN-R14del vs. 25.7±1.3 Hz in WT, p<0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital PCR and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9±2.3 Hz vs. 21.3±1.5 Hz; p<0.01). Conclusions: This study is the first to show that disruption of hPLN-R14del allele by AAV9- CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

Project description:Background: Phospholamban (PLN) is a tail-anchored sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+ 36 -reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Multi-protein complexes and higher-order supercomplexes, such as intercalated disk components and SER Ca+2 -cycling domains, underlie many critical cardiac functions, a subset of which may be disrupted by R14∆-PLN. Complexome profiling (CP) is a systems biology approach to characterize high molecular weight (MW) assemblies in biological samples. Most current CP data analysis strategies rely on external protein42 complex databases that are predominantly curated from immortalized or cancerous cell lines; thus, assemblies unique to specialized cell types such as cardiomyocytes, as well as higher-order supercomplexes that may have fluid stoichiometry/composition are significantly underrepresented. The requirement for computer programming expertise also presents a barrier to generalized adoption. Methods: We developed and applied a novel CP workflow to identify protein-complexes/supercomplexes disrupted in presymptomatic 28wk-old R14Δ/+ knockin mice hearts. Cardiac tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS)-based proteomics analysis. As expected, traditional database-dependent analysis offered only limited insights due to underrepresentation of cardiac-specific assemblies and higher-order supercomplexes. We thus developed a novel data analysis strategy (PERCOM) that does not rely upon external protein-complex databases and can, furthermore, be implemented on widely available spreadsheet software. Results: SEC-DIA-MS coupled with PERCOM analysis identified 296 proteins with disrupted MW-distribution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Mitochondrial alterations were confirmed at the level of global protein expression and metabolic function. Disruption of mitochondrial and ICD supercomplexes was confirmed in mice as young as 9wks of age. Conclusion: We present a novel CP workflow that is highly suited to the analysis of very high MW protein-complexes and supercomplexes in specialized tissue types. It includes a data analysis strategy that removes a computer programming resource barrier to general application. Our findings are among the first to show altered mitochondrial function as a hallmark of early-stage R14Δ-PLN, and identify disruption of the intercalated disk as a novel potential disease pathway. These molecular-level alterations target key machinery underlying cardiac energy generation and electromechanical connectivity, and may significantly contribute to the R14∆-PLN pathogenesis.

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Transcriptomic analyses of cardiomyopathy development in mice carrying the phospholamban p.(Arg14del) pathogenic variant, and comparison with ischemic heart disease

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